Mechanism Of Telomere Elongation Research Articles

Abstract C19: DNA demethylation induces upregulation of telomere repeat-containing RNA (TERRA) and downregulation of telomerase activity in human leukemia cells

Abstract Background and aim: Telomere elongation mechanisms have been proposed to be regulated by the epigenetic status of the telomeric chromatin. Telomere repeat-containing RNAs (TERRAs), which are RNAs that originate from telomeric DNA transcription, can associate with the telomeric chromatin, where they are proposed to function as negative regulators of telomere length based on their ability to act as potent inhibitors of telomerase in vitro. To elucidate the regulatory mechanism of telomere length in a chromatin-dependent manner, we analyzed factors that influence telomere/telomerase regulation in human leukemia cells treated with 5-azacytidine, a currently available demethylating agent. Methods: Reagents and cells: Human leukemia cell lines (U937, K562, and HL-60) were treated with 0.001 to 10 μM 5-azacytidine (Sigma) for 24, 48, and 72 h; 5-azacytidine was added every 24 h. The antiproliferative effect of 5-azacytidine was measured using the cell counting kit-8 (Wako Chemicals). Telomere, telomerase and TERRA: Telomere length and telomerase activity were measured as reported previously (Ohyashiki JH, Br J Cancer 2005; 92: 1942–1947). The telomeric localization of TERRA was determined by RNA-FISH with strand-specific telomeric DNA probes. Experiments were performed in denaturing conditions with or without RNase, and the number of TERRA signal-positive cells was measured. Methylation: Bisulfite genomic DNA sequencing was performed using primers designed to recognize both methylated and unmethylated forms of the p16/INK4a and p15/INK4b promoter regions. For the genome-wide screening of DNA methylation patterns, the Human Methylation27 BeadChip (Illumina) was used. Results: Demethylating effect of 5-azacytidine: The half maximal inhibitory concentration (IC50) of 5-azacytidine was 3.4 μM in U937, 3.6 μM in K562, and 1 μM in HL-60. Bisulfite genomic DNA sequencing revealed that the p15/INK4b promoter region was markedly demethylated in U937 cells after 72 h treatment with the IC50 dose of 5-azacytidine. Similarly, we noted genome-wide hypomethylation in 5-azacytidine-treated U937 cells using the Human Methylation27 BeadChip, whereas we noted only modest hypomethylation in 5-azacytidine-treated K562 and HL-60 cells. Upregulation of TERRA and telomerase inhibition: Based on the results obtained from the methylation analysis, we identified the localization of TERRA in 5-azacytidine-treated U937 and K562 cells. We observed 0∼2 dots corresponding to TERRA per cell in untreated U937 or K562 cells, and the signals were not detected in RNaseA-treated samples. In U937 cells, TERRA signals significantly increased in accordance with the decrease in telomerase activity. In contrast, neither an increase in TERRA signals nor a decrease in telomerase activity was observed in K562 cells. This indicates that the upregulation of TERRA and telomerase inhibition were closely related to genome-wide hypomethylation. Conclusion: There is emerging evidence for the clinical utility of demethylating agents, such as 5-azacytidine in myelodysplastic syndrome; however, the molecular mechanism of 5-azacytidine has not been fully elucidated. Our results indicate that 5-azacytidine induces the upregulation of TERRA, thereby inhibiting telomerase in U937 cells. Although there is still a long way to go before realizing the diagnostic and therapeutic uses of TERRA in leukemia patients, our study provided novel information on TERRA in the telomere and the telomerase biology of human leukemia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C19.

Human telomerase model shows the role of the TEN domain in advancing the double helix for the next polymerization step.

Telomerases constitute a group of specialized ribonucleoprotein enzymes that remediate chromosomal shrinkage resulting from the "end-replication" problem. Defects in telomere length regulation are associated with several diseases as well as with aging and cancer. Despite significant progress in understanding the roles of telomerase, the complete structure of the human telomerase enzyme bound to telomeric DNA remains elusive, with the detailed molecular mechanism of telomere elongation still unknown. By application of computational methods for distant homology detection, comparative modeling, and molecular docking, guided by available experimental data, we have generated a three-dimensional structural model of a partial telomerase elongation complex composed of three essential protein domains bound to a single-stranded telomeric DNA sequence in the form of a heteroduplex with the template region of the human RNA subunit, TER. This model provides a structural mechanism for the processivity of telomerase and offers new insights into elongation. We conclude that the RNADNA heteroduplex is constrained by the telomerase TEN domain through repeated extension cycles and that the TEN domain controls the process by moving the template ahead one base at a time by translation and rotation of the double helix. The RNA region directly following the template can bind complementarily to the newly synthesized telomeric DNA, while the template itself is reused in the telomerase active site during the next reaction cycle. This first structural model of the human telomerase enzyme provides many details of the molecular mechanism of telomerase and immediately provides an important target for rational drug design.

A two-step model for senescence triggered by a single critically short telomere

Telomeres protect chromosome ends from fusion and degradation. In the absence of a specific telomere elongation mechanism, their DNA shortens progressively with every round of replication, leading to replicative senescence. Here, we show that telomerase-deficient cells bearing a single, very short telomere senesce earlier, demonstrating that the length of the shortest telomere is a major determinant of the onset of senescence. We further show that Mec1p-ATR specifically recognizes the single, very short telomere causing the accelerated senescence. Strikingly, before entering senescence, cells divide for several generations despite complete erosion of their shortened telomeres. This pre-senescence growth requires RAD52 (radiation sensitive) and MMS1 (methyl methane sulfonate sensitive), and there is no evidence for major inter-telomeric recombination. We propose that, in the absence of telomerase, a very short telomere is first maintained in a pre-signalling state by a RAD52-MMS1-dependent pathway and then switches to a signalling state leading to senescence through a Mec1p-dependent checkpoint.

The Shelterin Protein TRF2 Inhibits Chk2 Activity at Telomeres in the Absence of DNA Damage

The shelterin complex [1] shapes and protects telomeric DNA from being processed as double strand breaks (DSBs) [2, 3]. Here we show that in human undamaged cells, a fraction of the kinase Chk2, a downstream target of ATM and mediator of checkpoint responses and senescence [4, 5], physically interacts with the shelterin subunit TRF2 and colocalizes with this complex at chromosome ends. This interaction, enhanced by TRF2 binding to telomeric DNA, inhibits the activation and senescence-induced function of Chk2 by a mechanism in which TRF2 binding to the N terminus of Chk2 surrounding Thr68 hinders the phosphorylation of this priming site. In response to radiation-induced DSBs, but not chromatin-remodelling agents, the telomeric Chk2-TRF2 binding dissociates in a Chk2 activity-dependent manner. Moreover, active Chk2 phosphorylates TRF2 and decreases its binding to telomeric DNA repeats, corroborating the evidences on the specific TRF2 relocalization in presence of DSBs [6]. Altogether, the capacity of TRF2 to locally repress Chk2 provides an additional level of control by which shelterin restrains the DNA damage response from an unwanted activation [6, 7] and may explain why TRF2 overexpression acts as a telomerase-independent oncogenic stimulus [8].

Separating the effects of telomere size from the mechanism of telomere elongation

Separating the effects of telomere size from the mechanism of telomere elongation

Drosophila telomere elongation.

Drosophila melanogaster has an unusual telomere elongation mechanism. Instead of short repeats that are synthesized by telomerase, long retrotransposons, HeT-A and TART, transpose to the ends of chromosomes. This mechanism generates tandem arrays of these elements at the chromosome ends, in which all elements are oriented with their oligo(A) tails towards the centromere. Structural features of HeT-A and TART elements may provide clues as to their transposition mechanism. Drosophila telomere length polymorphism is mainly due to terminal retrotransposon arrays that differ between chromosome tips and that change with time. In addition, stable terminal chromosome deletions can be generated that do not contain terminal HeT-A and TART arrays, suggesting that, unlike the equivalent terminal repeats in yeast and humans, the presence and length of terminal arrays in Drosophila may not be critical for cell cycle progression.

Telomere shortening and chromosomal instability abrogates proliferation of adult but not embryonic neural stem cells

Chromosome integrity is essential for cell viability and, therefore, highly proliferative cell types require active telomere elongation mechanisms to grow indefinitely. Consistently, deletion of telomerase activity in a genetically modified mouse strain results in growth impairments in all highly proliferative cell populations analyzed so far. We show that telomere attrition dramatically impairs the in vitro proliferation of adult neural stem cells (NSCs) isolated from the subventricular zone (SVZ) of telomerase-deficient adult mice. Reduced proliferation of postnatal neurogenic progenitors was also observed in vivo, in the absence of exogenous mitogenic stimulation. Strikingly, severe telomere erosion resulting in chromosomal abnormalities and nuclear accumulation of p53 did not affect the in vitro proliferative potential of embryonic NSCs. These results suggest that intrinsic differences exist between embryonic and adult neural progenitor cells in their response to telomere shortening, and that some populations of tissue-specific stem cells can bypass DNA damage check points.

Telomerase-independent mechanisms of telomere elongation.

The ends of linear chromosomes must be elongated in a DNA-replication-independent fashion. For chromosome end elongation the majority of eukaryotes use a specialized reverse transcriptase, telomerase, which adds a short, tandemly repeated DNA sequence motif to chromosome ends. Chromosome elongation can also be achieved, however, by mechanisms other than telomerase. Such elongation events have been detected under conditions where telomerase has been inactivated experimentally and in the few organisms that naturally lack telomerase. We will summarize current knowledge on these telomerase-independent elongation mechanisms in yeast and mammalian cells and will discuss in more detail the telomere elongation mechanism by retrotransposons in Drosophila melanogaster.

Repeated losses of TTAGG telomere repeats in evolution of beetles (Coleoptera).

We studied the occurrence of (TTAGG)n telomere repeats in 12 species of beetles, representing main lineages of the Coleoptera phylogenetic tree, by Southern hybridization and fluorescence in situ hybridization (FISH). In contrast to other insect orders, beetles were heterogeneous with respect to the occurrence of TTAGG repeats. In addition, the presence or absence of (TTAGG)n motif was irrespective of phylogenetic relationships. In the suborder Polyphaga, six species displayed positive hybridization signals. These were Silpha obscura, Agrilus viridis, Ampedus sanguineus, Stegobium paniceum, Oryzaephilus surinamensis, and Leptinotarsa decemlineata. Whereas negative signals were obtained in three polyphagan species, Geotrupes stercorarius, Thanasimusformicarius, and Sitophilus granarius. In the suborder Adephaga, the TTAGG sequence was present in one species, Graphoderus cinereus, and absent in two species, Orectochilus villosus and Pterostichus oblongopunctatus. We concluded that the telomerase-dependent (TTAGG)n motif had been repeatedly lost in different phylogenetic branches of Coleoptera and probably replaced with another mechanism of telomere elongation. This had to happen at least 5-6 times. The results suggest a predisposition or a backup mechanism of telomere maintenance in the genome of beetles that enabled them to make frequent evolutionary changes in the telomere composition.

The rate of terminal nucleotide loss from a telomere of the mosquito Anopheles gambiae.

Using a single copy pUChsneo transgene insertion at the Anopheles gambiae 2L telomere, this chromosome end was monitored by genomic Southern blots for forty-four mosquito generations. During this time, the chromosome end lost terminal nucleotides at an apparently constant rate of 55 bp/generation, which can be accounted for by incomplete DNA replication and does not imply exonuclease activity. No telomere elongation events were detected, suggesting that a previously described gene conversion event at this transgene does not occur very frequently. Moreover, no evidence for elongation by transposable elements was found, as described in Drosophila melanogaster. These results are consistent with the proposal that gene conversion between complex terminal satellite repeats that are present at natural telomeres, represents the major telomere elongation mechanism in A. gambiae. Such recombination events between repetitive sequences would occur more frequently than between the single copy pUChsneo transgene on the 2L homologues.

Telomerase Activity and Expression and Telomere Analysis in Situ in the Course of Treatment of Childhood Leukemias

ABSTRACTSamples of blood and marrow from children with leukemia were assayed for telomerase activity and expression on the day of diagnosis and during the course of chemotherapy. A strong correlation between either variables and clinical response was observed in most patients. A unique case was observed in which telomerase activity was only moderately increased on diagnosis; it gradually increased in the course of therapy, and a subsequent decrease occurred only after application of intensified therapy. This patient did not respond to therapy, his disease progressed, and he finally died during intensified therapy. In another patient, analysis of telomere lengths using dideoxy-PRINS revealed a single telomere expansion on a long arm of chromosome 4, suggesting involvement of a telomerase-independent mechanism of telomere elongation.

A telomeric satellite in Drosophila virilis and its sibling species.

Telomere elongation by telomerase is the most widespread mechanism among eukaryotes. However, alternative mechanisms such as homologous recombination between terminal satellite DNAs are probably used in lower dipteran insects and in some plants. Drosophila melanogaster uses the very unusual telomere elongation pathway of transposition of telomere-specific retrotransposable elements. The uniqueness of this telomere elongation mechanism raises the question of its origin. We, therefore, analyzed sequences located at telomeres of fairly distantly related Drosophila species, and in this paper we describe the characterization of complex satellite DNA sequences located at the telomeres of D. virilis and other species in the virilis group. We suggest an involvement of these DNA satellites in telomere elongation by homologous recombination similar to that found in lower dipterans. Our findings raise the possibility that telomere elongation by specific retrotransposons as found in D. melanogaster and its sibling species is a recent event in the evolution of dipteran insects.

A human breast cancer model for the study of telomerase inhibitors based on a new biotinylated-primer extension assay.

Telomerase is an RNA-dependent polymerase that synthesizes telomeric DNA (TTAGGG)n repeats. The overall goal of our work was to establish human cancer models that can be used to design clinical trials with telomerase inhibitors. The objectives of this study were (1) to set up a human breast cancer system that allows evaluation of the effects of telomerase inhibitors in cultured cells using a non-amplified telomerase assay and (2) to test this system using two drugs (cisplatin and TMPyP4) that affect the telomerase expression in breast cancer cells in culture. We first compared the telomerase activity in a variety of human breast cancer cell lines to that of other tumour types using a new biotinylated-primer extension assay. Our method, based on a non-amplified primer extension assay shows the direct incorporation of 32P-labelled nucleotides induced by telomerase on human telomeric primers. The 32P-dGTP labelled telomerase-extended 5′-biotinylated (TTAGGG)3 primer can subsequently be separated using streptavidin-coated magnetic beads. As compared to other non-amplified method, we showed that this procedure improved the characterization and the quantification of the banding pattern resulting from telomerase extension by reducing the radioactive background. Using this method, we observed that telomerase activity varies markedly in a panel of 39 human cancer cell lines. For example, MCF7 breast cancer cells in culture showed intermediate telomerase activity corresponding to 33.8 ± 3.4% of that of the HeLa cells (reference cell line). Similarly, the telomere length varied with each cell line (average: 6.24 ± 6.16). No correlation between the level of telomerase and telomere length was observed, suggesting that a high processivity is not required to maintain telomeres and that, in some cell lines, another mechanism of telomere elongation can maintain telomere length. From this study, we selected MCF7 and MX1 models that showed reproducible telomerase activity and a relatively limited telomere length for the testing of potential telomere–telomerase interacting agents. Using cisplatin and a new porphyrin-derived compound TMPyP4, we showed that our model was able to detect a down-regulation of the telomerase activity in MCF7 cells in culture and in a human MX1 tumour xenografts. Based on these results, a breast cancer model for evaluating telomerase and telomere interactive agents is proposed. © 1999 Cancer Research Campaign

Telomere maintenance without telomerase.

Telomeres are nucleoprotein structures at the ends of eukaryotic chromosomes that perform a number of vital functions. They allow a cell to distinguish between natural chromosome ends and chromosome breaks in order to delay the cell cycle and repair the broken end. Telomeres also compensate for the inability of DNA polymerase to replicate the chromosome completely. In most eukaryotes a special reverse transcriptase, telomerase, adds telomeric DNA repeats to the chromosome ends using an internal RNA template. However, evidence is accumulating for alternative elongation mechanisms in a variety of eukaryotes. In the yeast Saccharomyces cerevisiae, and possibly in humans, both of which normally use telomerase, a different mechanism can be used for chromosome length maintenance when telomerase is inactive or inactivated. Yeast apparently uses recombination for this purpose; the mechanism in humans is not known. Some insect and plant species, on the other hand, do not use telomerase as their primary mechanism for maintaining chromosome length. Drosophila makes use of specific retrotransposons for this purpose, while other dipterans use recombination. We summarize here the current knowledge of these alternative telomere elongation mechanisms.